<p>The detection and quantitation of protein-ligand binding interactions is important not only for understanding biological functions but also for the characterization of novel protein ligands. Because protein ligands can range from small molecules to other proteins, general techniques that can detect and quantitate the many classes of protein-ligand interactions are especially attractive. Additionally, the ability to detect and quantify protein-ligand interactions in complex biological mixtures would more accurately represent the protein-ligand interactions that occur in vivo, where differential protein expression and protein complexes can significantly affect a protein's ability to bind to a ligand of interest.</p><p> The work in this dissertation is focused on the development of new methodologies for the detection and measurement of protein-ligand interactions in complex mixtures using multiplex analyses. Methodologies for two types of multiplexed analyses of protein-ligand binding interactions are investigated here. The first type of multiplex analysis involves characterizing the binding of one protein target to many potential ligands, and the second type involves characterizing the binding of one ligand to many proteins. The described methodologies are derived from the SUPREX (stability of unpurified proteins from rates of H/D exchange) and SPROX (stability of proteins from rates of oxidation) techniques, which are chemical modification strategies that measure thermodynamic stabilities of proteins using a relationship between a protein's folding equilibrium and the extent of chemical modification. These two techniques were utilized in the development and application of several different experimental strategies designed to multiplex the analysis of protein-ligand interactions.</p><p> The first strategy that was developed involved a pooled compound approach for making SUPREX-based measurements of multiple ligands binding to a target protein. Screening rates of 6 s/ligand were demonstrated in a high-throughput screening project that involved the screening of two chemical libraries against human cyclophilin A (CypA), a protein commonly overexpressed in types of cancer. This study identified eight novel ligands to CypA with micromolar dissociation constants. Second, an affinity-based protein purification strategy was developed for the detection and quantitation of specific protein-ligand binding interactions in the context of complex protein mixtures. It involved performing SPROX in cell lysates and selecting the protein of interest using immunoprecipitation or affinity tag purification. A third strategy developed here involved a SPROX-based stable isotope labeling method for measuring protein-ligand interactions in multi-protein mixtures. This strategy was used in a proof-of-principle experiment designed to detect and quantify the indirect binding between yeast cyclophilin and calcineurin in a multi-component protein mixture. Finally, a quantitative proteomics platform was developed for the detection and quantitation of protein-ligand binding interactions on the proteomic scale. The platform was used to profile interactions of the proteins in a yeast cell lysate to several ligands, including the bioactive small molecules resveratrol and manassantin A, the cofactor nicotinamide adenine dinucleotide (NAD+), and two proteins, phosphoglycerate kinase (Pgk1) and pyruvate kinase (Pyk1). The above approaches should have broad application for use as discovery tools in the development of new therapeutic agents.</p>